99mTc-Labeling of HYNIC-Conjugated Cyclic RGDfK Dimer and

Publication Date (Web): February 19, 2008. Copyright © 2008 American Chemical Society. * To whom correspondence should be addressed: School of Health...
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Bioconjugate Chem. 2008, 19, 634–642

99m

Tc-Labeling of HYNIC-Conjugated Cyclic RGDfK Dimer and Tetramer Using EDDA as Coligand Jianjun Wang, Young-Seung Kim, and Shuang Liu* School of Health Sciences, Purdue University. Received November 15, 2007; Revised Manuscript Received January 1, 2008

In this study, EDDA (ethylenediamine-N,N′-diacetic acid) was used as the coligand for 99mTc-labeling of cyclic RGDfK conjugates: HYNIC-dimer (HYNIC ) 6-hydrazinonicotinamide; dimer ) E[c(RGDfK)]2) and HYNIC-tetramer (tetramer ) E{E[c(RGDfK)]2}2). First, HYNIC-dimer was allowed to react with 99mTcO4- in the presence of excess tricine and stannous chloride to form the intermediate complex [99mTc(HYNIC-dimer)(tricine)2], which was then allowed to react with EDDA to afford [99mTc(HYNIC-dimer)(EDDA)] with high yield (>90%) and high specific activity (∼8.0 Ci/µmol). Under the same radiolabeling conditions, the yield for [99mTc(HYNIC-tetramer)(EDDA)] was always 25% ID/g) over the 2 h study period, probably due to the negative charges on the TPPTS coligand (31). EDDA (EDDA ) ethylenediamine-N,N′-diacetic acid) has been used for 99mTc-labeling of the HYNIC-conjugated somatostatin analogues (36–38) and RGD peptide (39). It was found that [99mTc(HYNIC-RGD)(EDDA)] had a high tumor uptake, along with the rapid renal excretion, and low uptake in the liver and muscle. The tumor uptake studies also showed specific targeting of integrin Rvβ3 in the mice bearing small cell lung tumors. All tumors could be readily visualized by scintigraphic imaging. It was claimed that [99mTc(HYNIC-RGD)(EDDA)] had tumor/organ ratios comparable to those of [18F]Galato-RGD (39). These promising results lead us to explore the use of EDDA as the coligand for 99mTc-labeling of the HYNIC-dimer and HYNIC-tetramer conjugates. The main objective is to demonstrate whether EDDA is useful for minimizing the kidney uptake of 99mTc-labeled cyclic RGD dimer and tetramer. In this study, we used EDDA as coligand for 99mTc-labeling of both HYNIC-dimer and HYNIC-tetramer. A mixed-ligand experiment was used to determine the number of EDDA ligands bonding to the 99mTc-HYNIC core. The athymic nude mice bearing U87MG human glioma xenografts were used to evaluate the impact of EDDA coligand on biodistribution properties of the 99mTc-labeled HYNIC-dimer and HYNIC-tetramer. In addition, we also examined the relationship between the size and tumor uptake of [99mTc(HYNIC-dimer)(EDDA)], [99mTc

10.1021/bc7004208 CCC: $40.75  2008 American Chemical Society Published on Web 02/19/2008

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Tc-Labeling of Cyclic RGDfK Conjugates

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Figure 1. HYNIC-conjugated cyclic RGDfK dimer (HYNIC-dimer) and tetramer (HYNIC-tetramer).

Figure 2. Typical radio-HPLC chromatograms (method 1) of [99mTc (HYNIC-dimer)(tricine)2] (top) and [99mTc(HYNIC-dimer) (EDDA)] (bottom).

(HYNIC-tetramer)(EDDA)], and [99mTc(HYNIC-tetramer) (tricine)(TPPTS)].

EXPERIMENTAL SECION Materials. Ethylenediamine-N,N′-diacetic acid (EDDA) and tricine were purchased from Sigma/Aldrich (St. Louis, Mis-

souri). Pentapeptide c(RGDfK) was purchased from Peptides International, Inc. (Louisville, Kentucky). HYNIC-E[c(RGDfK)]2 (HYNIC-dimer), HYNIC-E{E[c(RGDfK)]2}2 (HYNIC-tetramer), and [99mTc(HYNIC-tetramer)(tricine)(TPPTS)]werepreparedaccordingtotheliteratureprocedures(29,31). N,N′-Bis(benzyl)ethylenediamine-N,N′-diacetic acid (Bn2EDDA) was prepared according to the literature method (40). Na99mTcO4 was obtained from a commercial DuPont Pharma 99Mo/99mTc generator (N. Billerica, MA). Instruments and Methods. The radio-HPLC method (method 1) used the LabAlliance HPLC system equipped with a β-ram IN-US detector and Zorbax C18 column (4.6 mm × 250 mm, 300 Å pore size). The flow rate was 1 mL/min. The mobile phase was isocratic with 90% solvent A (25 mM NH4OAc buffer, pH ) 5.0) and 10% solvent B (acetonitrile) at 0–2 min, followed by a gradient mobile phase going from 10% solvent B at 2 min to 15% solvent B at 5 min and to 20% solvent B at 20 min. Method 2 was almost identical to method 1 except that the gradient mobile phase was used from 20% solvent B at 5 min and to 40% solvent B at 20 min. Synthesis of [99mTc(HYNIC-dimer)(EDDA)]. To a 5 cc vial containing tricine (20 mg in 1 mL of succinate buffer (pH ) 5) was added 0.2 mL of HYNIC-dimer solution (100 µg/ mL), 0.3 mL of 99mTcO4- solution in saline, and 25 µL SnCl2 solution (1 mg/mL) in 1 N HCl. The vial containing the reaction mixture was heated at 100 °C for 20 min. A sample of the resulting solution was analyzed by HPLC (method 1). To the vial above was added 0.5 mL of EDDA solution (10 mg/mL, pH ∼ 7.4). The resulting mixture was heated at 100 °C for 20 min. After cooling to room temperature, a sample from the solution was analyzed by HPLC (method 1). The radiochemical purity (RCP) was >95%. The HPLC retention time was 13.2 min.

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Figure 3. Radio-HPLC chromatograms (method 1) of [99mTc(HYNIC-dimer)(Bz2EDDA)] (top), [99mTc(HYNIC-dimer)(EDDA)] (middle), and the reaction solution (bottom) containing [99mTc(HYNIC-dimer)(Bz2EDDA)] and [99mTc(HYNIC-dimer)(EDDA)]. The presence of only two sets of peaks at 13.5 and 17.0 min from [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNICdimer)(Bn2EDDA)], respectively, clearly demonstrated that there is only one EDDA bonded to the 99mTc-HYNIC core. Table 1. Stability Data for [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] in the Presence of Cysteine and Histidine (1 mg/mL in 25 mM phosphate buffer, pH ) 7.5) time post purification 0.5 h 1.0 h 2.0 h 4.0 h 6.0 h

RCP (%) in histidine solution RCP (%) in cysteine solution (1 mg/mL, pH ) 7.4) (1 mg/mL, pH ) 7.4) dimer

tetramer

dimer

tetramer

100 100 99 97 95

99 99 98 98 97

100 100 98 98 95

99 99 98 96 96

Synthesis of [99mTc(HYNIC-dimer)(Bn2EDDA)]. To a clean 5 cc vial containing tricine (20 mg in 1 mL succinate buffer, pH ) 5) was added 0.2 mL of HYNIC-dimer solution (100 µg/mL), 0.3 mL of 99mTcO4- solution in saline, and 25 µL SnCl2 solution (1 mg/mL) in 1 N HCl. The vial containing the reaction mixture was heated at 100 °C for 20 min. To the vial was added 0.5 mL of Bn2EDDA solution (10 mg/mL, pH ∼ 7.4). The vial was heated at 100 °C for another 20 min. After cooling to room temperature, a sample from the solution was analyzed by radio-HPLC (method 1). The RCP was >95%. The HPLC retention time was 17.2 min.

Synthesis of [99mTc(HYNIC-tetramer)(EDDA)]. To a 5 cc vial containing tricine (20 mg in 1 mL succinate buffer, pH ) 5) was added 0.2 mL of HYNIC-tetramer solution (100 µg/ mL), 0.3 mL of 99mTcO4- solution, and 25 µL SnCl2 solution (1 mg/mL) in 1 N HCl. The vial containing the reaction mixture was heated at 100 °C for 20 min. To the vial above was added 0.5 mL of EDDA solution (10 mg/mL, pH ∼ 7.4). The resulting mixture was heated at 100 °C for 20 min. After cooling to room temperature, a sample from the solution was analyzed by radioHPLC (method 2). The RCP was ∼65%. The HPLC retention time was 15.2 min. Doses Preparation and Solution Stability. [99mTc(HYNICdimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] were prepared. Doses were prepared by dissolving the purified radiotracer in saline to give a concentration of 20 µCi/mL for biodistribution studies and 1.0 mCi/mL for metabolism studies. Each animal was injected with 0.1 mL of the dose solution. For cysteine and histidine challenging experiments, the radiotracer was purified by HPLC. Volatiles in mobile phases were removed under vacuum. The residue was dissolved in the cysteine or histidine solution (1 mg/mL, pH ) 7.4). Samples of the resulting solution were analyzed by radio-HPLC (method 1 for [99mTc(HYNIC-dimer)(EDDA)] and method 2 for [99mTc(HYNIC-tetramer)(EDDA)]) at 0, 2, 4, and 6 h. Determination of Log P Values. Log P values were determined using the following procedure: The radiotracer was purified by HPLC. Volatiles were removed completely under vacuum. The residue was dissolved in a equal volume (3 mL/3 mL) mixture of n-octanol and 25 mM phosphate buffer (pH ) 7.4). After stirring vigorously for ∼20 min, the mixture was centrifuged at a speed of 8000 rpm for 5 min. Samples (in triplets) from both n-octanol and aqueous layers were counted in a gamma counter (Perkin-Elmer Wizard - 1480). The log P value was measured three different times and reported as an average of three different measurements plus the standard deviation. The log P values were -3.59 ( 0.11 and -3.26 ( 0.08 for [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNICtetramer)(EDDA)], respectively. Mixed-Ligand Experiment. To a clean 5 cc vial containing tricine (20 mg in 1 mL of succinate buffer, pH ) 5) was added 0.2 mL of HYNIC-dimer solution (100 µg/mL), 0.3 mL of 99m TcO4- solution in saline, and 25 µL SnCl2 solution (1 mg/ mL) in 1 N HCl. The reaction mixture was heated at 100 °C for 20 min. To the mixture above was added 0.5 mL of the solution containing EDDA (10 mg/mL, pH ∼7.4) and Bn2EDDA (10 mg/mL, pH ∼7.4). The resulting mixture was heated at 100 °C for another 20 min. After cooling to room temperature, a sample of the resulting solution was analyzed by the radio-HPLC (method 1). Animal Model. Biodistribution studies were performed according to the literature procedures (30). These studies were carried out using the athymic nude mice bearing U87MG human glioma xenografts in compliance with NIH animal experiment guidelines (Principles of Laboratory Animal Care, NIH Publication No. 86–23, revised 1985). The animal protocol for the animal studies has been approved by the Purdue University Animal Care and Use Committee (PACUC). U87MG human glioma cells were grown at 37 °C in Minimal Essential Medium (Alpha Modification) containing 3.7 g of sodium bicarbonate/ L, 10% fetal bovine serum v/v, in a humidified atmosphere of 5% carbon dioxide. Female athymic nu/nu mice were purchased from Harlan (Indianapolis, IN) at 4–5 weeks of age. Each mouse was orthotopically implanted with 5 × 106 U87MG human glioma cells into the mammary fat pad. Four to five weeks after inoculation, animals with tumors in the range 0.1–1.5 g were used for biodistribution studies.

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Table 2. Biodistribution Data of [99mTc(HYNIC-dimer)(EDDA)] in the Athymic Nude Mice Bearing the U87MG Human Glioma Xenografts (n ) 5 for each time point) unless Specified organ (% ID/gram)

5 min

30 min

60 min (n ) 8)

120 min (n ) 6)

blood brain heart intestine kidney liver lungs muscle spleen tumor tumor/blood tumor/brain tumor/liver tumor/lung tumor/muscle

4.41 ( 0.48 0.25 ( 0.05 3.20 ( 0.34 7.58 ( 2.05 25.25 ( 5.64 5.29 ( 0.29 6.36 ( 0.91 3.85 ( 0.60 2.30 ( 0.19 4.17 ( 2.35 1.47 ( 0.88 30.79 ( 18.32 1.35 ( 0.59 1.11 ( 0.43 1.85 ( 0.80

1.56 ( 0.33 0.15 ( 0.02 1.62 ( 0.22 7.55 ( 3.43 11.70 ( 2.25 4.03 ( 0.55 4.32 ( 0.30 2.60 ( 1.14 1.43 ( 0.45 5.44 ( 1.54 4.45 ( 1.29 46.37 ( 10.37 1.69 ( 0.40 1.58 ( 0.28 2.78 ( 0.60

1.17 ( 0.12 0.15 ( 0.03 1.33 ( 0.17 4.63 ( 1.18 8.42 ( 1.33 3.97 ( 0.39 3.32 ( 0.27 2.66 ( 0.46 1.26 ( 0.30 4.07 ( 2.02 5.28 ( 0.92 36.56 ( 7.43 1.38 ( 0.40 1.64 ( 0.42 2.07 ( 0.61

0.57 ( 0.25 0.10 ( 0.02 0.86 ( 0.15 3.03 ( 0.54 5.97 ( 0.94 2.98 ( 0.48 2.10 ( 0.36 2.02 ( 0.23 0.77 ( 0.11 6.44 ( 1.76 12.55 ( 5.33 66.33 ( 23.53 2.21 ( 0.70 3.10 ( 0.85 3.24 ( 1.02

Table 3. Biodistribution Data of [99mTc(HYNIC-tetramer)(EDDA)] in the Athymic Nude Mice Bearing the U87MG Human Glioma Xenografts (n ) 5 for Each Time Point unless Specified) organ

5 min

30 min

60 min

120 min (n ) 6)

blood brain heart intestine kidney liver lungs muscle spleen tumor tumor/blood tumor/brain tumor/liver tumor/lung tumor/muscle

3.16 ( 0.78 0.28 ( 0.03 4.10 ( 0.53 21.38 ( 4.86 26.69 ( 2.48 7.56 ( 1.48 6.07 ( 0.86 2.11 ( 0.37 3.97 ( 0.80 4.39 ( 1.00 1.43 ( 0.30 15.44 ( 1.98 0.59 ( 0.17 0.75 ( 0.27 2.13 ( 0.62

1.47 ( 0.32 0.18 ( 0.04 2.87 ( 0.91 14.47 ( 2.08 17.84 ( 2.82 5.67 ( 1.23 4.67 ( 1.00 1.66 ( 0.27 3.28 ( 1.10 5.13 ( 3.10 3.38 ( 2.70 26.44 ( 6.78 0.58 ( 0.22 0.98 ( 0.62 2.77 ( 0.07

0.54 ( 0.11 0.13 ( 0.02 1.72 ( 0.22 8.01 ( 1.31 11.01 ( 0.88 4.22 ( 0.70 3.76 ( 0.58 0.96 ( 0.15 2.77 ( 0.73 4.70 ( 0.82 8.98 ( 1.35 34.82 ( 6.11 1.09 ( 0.31 1.22 ( 0.19 4.89 ( 0.88

0.45 ( 0.18 0.11 ( 0.02 1.51 ( 0.39 8.27 ( 1.92 9.54 ( 2.23 4.60 ( 0.82 3.23 ( 0.99 0.92 ( 0.26 2.44 ( 0.35 6.57 ( 1.63 15.89 ( 4.77 60.43 ( 7.53 1.47 ( 0.50 2.11 ( 0.44 7.43 ( 2.03

Biodistribution Protocol. 20 tumor-bearing mice (20–25 g) were anesthetized with intraperitoneal injection of a mixture containing Ketamine (40–100 mg/kg) and Xylazine (2–5 mg/ kg). Once the animal was in the surgical plane of anesthesia, as noted by the lack of pain response, the radiotracer (∼2 µCi) dissolved in 0.1 mL saline was administered via tail vein. Five animals were sacrificed by sodium pentobarbital overdose (100 mg/kg) at 5, 30, 60, and 120 min postinjection (p.i.). Blood samples were withdrawn from the heart through a syringe. Organs (tumor, brain, eyes, spleen, lungs, liver, kidneys, muscle, and intestine) were excised, washed with saline, weighed, and counted on a gamma counter. The organ uptake was calculated as a percentage of the injected dose per organ (% ID/organ) and a percentage of the injected dose per gram of organ tissue (% ID/g). The biodistribution data and target-to-background (T/ B) ratios are reported as an average plus the standard variation. Comparison between two different radiotracers was also made using the one-way ANOVA test (GraphPad Prism 5.0, San Diego, CA). The level of significance was set at p < 0.05. Metabolism. Two normal mice were used for metabolism studies. Each mouse was administered the radiotracer at a dose of 100 µCi/mouse. The urine samples were collected at 30 min and 2 h p.i. by manual void, and were mixed with equal volume of acetonitrile. The mixture was centrifuged at 8000 rpm. The supernatant was collected and filtered through a 0.20 µm MillexLG syringe driven filter unit. The filtrate was analyzed by radioHPLC (method 1 for [99mTc(HYNIC-dimer)(EDDA)] and method 2 for [99mTc(HYNIC-tetramer)(EDDA)]). The feces samples were collected at ∼120 min p.i. The sample was suspended in a mixture of 50% acetonitrile aqueous solution, and the resulting mixture was vortexed for 5–10 min. After

centrifuging, the supernatant was collected and passed through a 0.20 µm Millex-LG syringe driven filter unit. The filtrate was analyzed by radio-HPLC.

RESULTS Radiochemistry. [99mTc(HYNIC-dimer)(EDDA)]and[99mTc(HYNIC-tetramer)(EDDA)] (Figure 1) were prepared in two steps. HYNIC-dimer was first allowed to react with 99mTcO4in the presence of excess tricine and stannous chloride to form the [99mTc(HYNIC-dimer)(tricine)2] intermediate, which was then reacted with EDDA to form [99mTc(HYNIC-dimer)(EDDA)]. [99mTc(HYNIC-tetramer)(EDDA)] was prepared in the same fashion using HYNIC-tetramer. Attempts to prepared [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] in one step by directly reacting the HYNIC-dimer or HYNIC-tetramer with 99mTcO4- in the presence of EDDA and stannous chloride were not successful due to their low yield (90% using 20 µg of HYNIC-dimer (∼1.2 × 10-8 mol) and 20 mCi of 99mTcO4- (∼1.5 × 10-10 mol of 99m Tc/99Tc for 24 h generator). The specific activity was ∼8.0 Ci/µmol for [99mTc(HYNIC-dimer)(EDDA)]. In contrast, the radiolabeling yield was always 6 h, suggesting that they are intact before being injected into animals. Biodistribution Characteristics. The athymic nude mice bearing U87MG glioma xenografts were used to study biodistribution properties of [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)]. Both [99mTc(HYNICdimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] were prepared, and purified by HPLC to remove excess “unlabeled” HYNIC conjugate that may compete with the radiotracer in binding to the integrin Rvβ3 expressed on tumor cells and in normal tissues. Biodistribution data for [99mTc(HYNIC-dimer) (EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] are summarized in Tables 2 and 3, respectively. In general, [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] have a rapid blood clearance. Their tumor uptake was comparable within the experimental error over the 2 h study period. Their lung uptake also compared favorably during the same study period. The liver uptake of [99mTc(HYNIC-dimer)(EDDA)] was 5.29 ( 0.29% ID/g and 2.98 ( 0.48% ID/g at 5 and 120 min p.i., respectively, and was significantly lower (p < 0.01) than that of [99mTc(HYNICtetramer)(EDDA)] (7.56 ( 1.48% ID/g and 4.60 ( 0.82% ID/g at 5 and 120 min p.i., respectively). The initial kidney uptake of [99mTc(HYNIC-dimer)(EDDA)] (25.25 ( 5.64% ID/g at 5 min p.i.) was almost identical to that of [99mTc(HYNICtetramer)(EDDA)] (26.69 ( 2.48% ID/g at 5 min p.i.); but this similarity disappeared at >30 min p.i. The kidney uptake of [99mTc(HYNIC-dimer)(EDDA)] was 11.70 ( 2.25% ID/g and 5.97 ( 0.94% ID/g at 30 and 120 min p.i., respectively, while the kidney uptake of [99mTc(HYNIC-tetramer)(EDDA)] was 17.84 ( 2.82% ID/g at 5 min p.i. and 9.54 ( 2.23% ID/g at 120 min p.i. The muscle uptake of [99mTc(HYNIC-dimer)(EDDA)] (3.85 ( 0.60% ID/g and 2.02 ( 0.23% ID/g at 5 and 120 min p.i., respectively) was significantly higher than that of [99mTc(HYNIC-tetramer)(EDDA)] (2.11 ( 0.37% ID/g and 0.92 ( 0.26% ID/g at 5 and 120 min p.i., respectively). Figure 4 compares the organ uptake (% ID/g) of [99mTc(HYNIC-dimer)(EDDA)], [99mTc(HYNIC-dimer)(tricine)(TPPTS)], [99mTc(HYNIC-tetramer)(EDDA)], and [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] in the liver, kidneys, and lungs. The biodistribution data for [99mTc(HYNIC-dimer)(tricine)(TPPTS)] and [99mTc(HYNIC-tetramer)(tricine) (TPPTS)] were obtained from our previous studies in the same tumor-bearing animal model (29, 31). Replacing tricine/TPPTS with EDDA did not change the uptake of the 99mTc-labeled HYNIC-dimer in kidneys, liver, or lungs. [99mTc(HYNICtetramer)(EDDA)] also had the liver and lung uptake very similar to that of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)]; but it had a much lower kidney uptake than [99mTc(HYNICtetramer)(tricine)(TPPTS)] over the 2 h period (Figure 4). During biodistribution studies, we noticed a large variation in the tumor uptake for both [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)], and their tumor uptakes were largely dependent upon the tumor size. Similar behavior was reported for the 99mTc-labeled cyclic RGDfK monomer (11). To further clarify this relationship, we added extra tumorbearing mice into the 60 and 120 min groups for both radiotracers. We also performed a biodistribution study on [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] at 120 min p.i. The biodistribution data at 60 and 120 min were used for all three

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Figure 5. The relationship between the tumor size and tumor uptake (expressed as (% ID/g) for [99mTc(HYNIC-dimer)(EDDA)] at 60 min p.i. (A: n ) 8) and 120 min p.i. (B: n ) 5), [99mTc(HYNIC-tetramer)(EDDA)] at 120 min p.i. (C: n ) 6), and [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] at 120 min p.i. (D: n ) 8) in the athymic nude mice bearing the U87MG human glioma xenografts.

radiotracers to minimize the interference from their blood activity. Figure 5 shows the inverse linear relationship between the tumor size and tumor uptake (expressed as % ID/g) of [99mTc(HYNIC-dimer)(EDDA)] at 60 min (A) and 120 min (B) p.i., [99mTc(HYNIC-tetramer)(EDDA)] (C), and [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] (D) at 120 min p.i. in the same model. It is clear that the radiotracer tumor uptake (% ID/g) decreases as the tumor size increases. Smaller tumors (5% ID/g) that large tumors (>1.0 g) regardless of the identity of radiotracer. Metabolic Properties. Normal athymic mice were used to examine metabolic stability of [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)]. Since >85% of [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNICtetramer)(EDDA)] was excreted from the renal and hepatobiliary routes by 2 h p.i., we used radio-HPLC to analyze urine and feces samples from the mice administered with the radiotracer (∼100 µCi/mouse). Figure 6 shows radio-HPLC chromatograms of [99mTc(HYNIC-dimer)(EDDA)] (left) and [99mTc(HYNICtetramer)(EDDA)] (right) in saline (A), in the urine at 30 min p.i. (B), in the urine at 120 min p.i. (C), and in the feces at 120 min p.i. (D). There were no metabolites detected in the urine at 30 and 120 min p.i. for both radiotracers, indicating that they have a high metabolic stability during excretion from the renal system. Only 35% of [99mTc(HYNIC-dimer)(EDDA)] and 15% of [99mTc(HYNIC-tetramer)(EDDA)] remained intact in the feces samples, suggesting that they

underwent significant metabolism during excretion from the hepatobiliary system.

DISCUSSION Recently, Decristoforo et al. (39) reported the 99mTc-labeled cyclic RGD monomer, HYNIC-c(RGDyK) (HYNIC-RGD). It was found that HYNIC-RGD could be labeled with 99mTc in high specific activity (∼100 GBq/µmol or 2.7 Ci/ µmol) using EDDA as coligand. Despite the fact that [99mTc(HYNICRGD)(EDDA)] had a high tumor uptake, along with the rapid renal excretion, and low uptake in the liver and muscle, it is not known about the composition of 99mTc radiotracer even though the LC-MS data suggested that there were two EDDA ligands binding to the 99mTc-HYNIC core (41). In this study, we used EDDA as the coligand to prepare [99mTc(HYNICdimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)]. The specific activity was ∼300 GBq/µmol or 8 Ci/µmol for [99mTc(HYNIC-dimer)(EDDA)] using the two-step radiolabeling procedure. Both [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] have very high in Vitro solution stability (Table 1). There are several important findings from this study. The results from a mixed-ligand experiment clearly demonstrate that there is only one EDDA bonding to the 99mTc-HYNIC core in [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] (Figure 3). To our surprise, [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] have almost identical tumor uptake (Tables 2 and 3). There is no advantage of the 99mTc-labeled HYNIC-tetramer over its dimeric analogue when EDDA is used as the coligand. This seems to contradict

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Figure 6. Typical radio-HPLC chromatograms of the HPLC-purified [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] in saline before injection, in urine at 30 min p.i., in urine at 120 min p.i., and in feces at 120 min p.i. Each mouse was administered with ∼ 100 µCi radiotracer. Two normal mice were used for each radiotracer.

the results obtained from 111In(DOTA-tetramer) (33), 64 Cu(DOTA-tetramer) (26, 28), and [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] (29, 31) over their dimeric analogues. There is no clear explanation for this observation. Replacing tricine/ TPPTS with EDDA did not significantly change the uptake of the 99mTc-labeled HYNIC-dimer in kidneys, liver, and lungs; but it did result in a significantly lower kidney uptake for [99mTc (HYNIC-tetramer)(EDDA)] due to its faster renal excretion (Figure 4) than [99mTc(HYNIC-tetramer)(tricine)(TPPTS)].

Apparently, coligands have a significant impact on excretion kinetics of the 99mTc-labeled HYNIC-tetramer. It is very important to note that the tumor growth rate varies significantly even though each mouse receives the same number of tumor cells for the same inoculation time. The radiotracer tumor uptake decreases as the tumor size increases in a linear fashion. The smaller the tumors are, the higher the tumor uptake (% ID/g) is regardless of the identity of radiotracer (Figure 5). Since Chen and co-workers have elegantly demonstrated that

99m

Tc-Labeling of Cyclic RGDfK Conjugates

the radiotracer tumor uptake is well-correlated with the integrin Rvβ3 expression levels in tumors (42), we believe that the reduced radiotracer uptake (% ID/g) might be related to the integrin Rvβ3 density. The radiotracer tumor uptake expressed as %ID reflects the total number of integrin Rvβ3 receptors expressed on both tumor cells and endothelial cells, while the tumor uptake expressed as %ID reflects the integrin Rvβ3 density. In small tumors, the microvessel density and the integrin Rvβ3 density are high. The tumor uptake expressed as % ID/g is high (Figure 5) even though the radiotracer tumor uptake expressed as %ID is low (Supporting Information). As the tumor grows, the total number of integrin Rvβ3 receptors expressed on tumor cells becomes larger, and the tumor uptake expressed as %ID increases (Supporting Information). In contrast, the microvessel density decreases because of maturity of blood vessels, and the integrin Rvβ3 density also decreases due to larger interstitial space and higher collagen concentrations (43). In addition, part of the tumor tissue may also become necrotic, which may also lead to lower the integrin Rvβ3 density in larger tumors. As a result, the radiotracer tumor uptake expressed as % ID/g in larger tumors is significantly lower than that of smaller ones (Figure 5). Thus, it is important to select the mice bearing tumors of the same or similar size for biological evaluations. This may become more important when comparing radiotracers for their tumor uptake and tumor-targeting capability. However, this will, to a certain degree, compromise the “random nature” of the evaluation process. Radiolabeled small peptides often accumulate in kidneys and liver (44). Extensive metabolic degradation was observed for the 99mTc-labeled cyclic RGDfK monomer (10), dimer (29), and tetramer (31), and the 64Cu-labeled cyclic RGDfK tetramer in kidneys and urine samples (28). In this study, [99mTc(HYNICdimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] (Figure 6) show very high metabolic stability during their excretion from the renal system and significant metabolism during their excretion from the hepatobiliary system, probably due to rapid enzymatic degradation of the cyclic RGD peptide and/or the 99m Tc chelate. The difference in the metabolic fate between [99mTc(HYNIC-tetramer)(EDDA)] described in this study and [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] (31) seems to be related to coligands (EDDA vs tricine/TPPTS) in the Tc chelate.

CONCLUSION In summary, we have described the synthesis and evaluation of [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNICtetramer)(EDDA)] as potential radiotracers for tumor imaging using athymic nude mice bearing the U87MG glioma xenografts. The key findings of this study are as follows: (1) there is only one EDDA binding to the 99mTc-HYNIC core; (2) the use of EDDA as coligand to replace tricine/TPPTS does not significantly change the uptake of the 99mTc-labeled HYNIC-dimer in liver, kidneys, and lungs; but it results in a significantly lower kidney uptake for the 99mTc-labeled HYNIC-tetramer due to faster radiotracer renal excretion; and (3) the radiotracer tumor uptake decreases as the tumor size increases in a linear fashion.

ACKNOWLEDGMENT We would like to thank Dr. Sulma I. Mohammed, the Director of Purdue Cancer Center Drug Discovery Shared Resource, Purdue University, for her assistance with the tumor-bearing animal model. This work is supported, in part, by Purdue University and research grants: R01 CA115883 A2 (S.L.) from National Cancer Institute (NCI), BCTR0503947 (S.L.) from Susan G. Komen Breast Cancer Foundation, R21 EB00341902 (S.L.) from National Institute of Biomedical Imaging and

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Bioengineering (NIBIB) and R21 HL083961-01 from National Heart, Lung, and Blood Institute (NHLBI). Supporting Information Available: Additional figure. This material is available free of charge via the Internet at http:// pubs.acs.org.

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